Increasing genomic and cytological evidence suggests that where a gene is located within the nucleus and how it is folded within the interphase chromosome may produce comparable increases in a gene's transcriptional output as the initial binding of transcription factors to promoter sequences. Our long-term goals are to determine the intranuclear positioning and large-scale chromatin structure of specific gene loci, to identify the cis and trans determinants of this positioning and folding, and to determine the functional significance of this level of chromatin organization. Our hypothesis is that gene positioning and large-scale chromatin folding create a global chromatin environment whose influence on transcriptional regulation of certain genes is quantitatively is at least as large as the influence of local cis regulatory elements. Our goals for the next funding period will be to: 1) develop a new genomic method for measuring actual large-scale chromatin compaction; 2) determine the functional connections between gene positioning to the nuclear lamina and speckles and large-scale chromatin compaction with gene regulation; 3) dissect the relationships between large-scale chromatin folding, chromatin domain epigenetic states, and nuclear compartmentalization with each other and with transcriptional regulation. This work builds on several key advances during the last funding period, including: a) Development of our novel TSA-Seq method for genome-wide mapping of intranuclear gene positioning; b) Demonstration of deterministic positioning of >50% of the most highly expressed genes near nuclear speckles; c) Demonstration of long-range, directed movement of Hsp70 transgenes loci to nuclear speckles upon heat-shock; d) Demonstration of a tight correlation between speckle association and full Hsp70 transgene activation; e) Reconstitution of large chromatin domains with distinct nuclear compartmentalization, epigenetic, and large-scale chromatin condensation states and identification of cis and trans factors which determine these states. Together these advances enable this proposal's unique ?Divide-and-Conquer? experimental approach in which we identify trans factors regulating gene positioning and/or large-scale chromatin compaction of particular gene loci and then use genomic methods to survey the genome-wide relevance of these trans factors to gene positioning, large-scale chromatin compaction, and gene regulation. Through this research we will gain a better understanding of gene regulation and how to manipulate gene expression for possible future therapeutic approaches.